The unicellular eukaryote, Tetrahymena thermophila, has two kinds of nuclei: The diploid micronucleus, the germ-line nucleus, which can undergo meiosis; the 45c macronucleus, the somatic nucleus, which is replaced each sexual generation from micronuclear products. The purpose of the proposed research is to investigate the molecular events underlying the development and differentiation of the macronucleus. During this process genome alterations occur, including gene loss, and excess DNA is generated. We plan to determine whether DNA sequences are eliminated only at one developmental stage or more gradually, if the same sequences are always involved, and if the excess DNA generated includes sequences destined to be eliminated from the genome. We will also determine whether genome alteration involves fragmentation or recombination of chromosomes, which DNA sites are involved, and whether segments derived from different chromosomes recombine at these sites. We will use the gene cloning techniques and select particular sequences from the micronucleus and macronucleus, and use the selected cloned sequences in renaturation and hydridization experiments with DNA obtained from nuclei at various stages and states of differentiation. These studies should contribute towards understanding how alterations in DNA molecules occur in normal cells of eukaryotic organisms during development and differentiation.